The microtubule cytoskeleton acts as both the neuronal highway system and the signposts. Even small alterations in microtubule motor or organizing proteins can result in neurodegeneration as these long cells rely on transport from the cell body to axons and dendrites to function for a lifetime. While neuronal microtubules are critically important for neuronal health, many of the basic mechanisms that organize them have not yet been identified. In this proposal we focus on elucidating mechanisms that control dendrite microtubule polarity. Unlike axons, dendrites have large numbers of minus-end-out microtubules that direct dendrite-specific proteins and organelles to this compartment. As minus-end-out microtubules specify dendrite identity and are poorly understood we will maximize our impact by focusing on their generation and regulation. We previously found that dendrite branch points are hubs of microtubule control and house: 1) machinery that steers microtubules towards the cell body, and 2) nucleation sites. It was proposed that Golgi outposts localize nucleation sites to dendrites, however we have shown this is not the case. Using the microtubule steering machinery as a starting point, we have identified several sets of proteins, including heterotrimeric G proteins and branched actin regulators that play a role in branch point targeting. In Aim 1, we will determine which of these protein sets also positions nucleation sites, and how they influence overall microtubule polarity. In Aim 2 we will focus on the part of the dendrite beyond the branch point. Although nucleation sites are concentrated at branch points, microtubule minus ends are found even near dendrite tips. We will explore how they might get there to identify new players and mechanisms in microtubule control. For example, we can observe growing minus ends in dendrites and hypothesize these may allow population of distal dendrites with microtubules. In the last Aim of the proposal we will jump into new territory and take maximum advantage of our model system and our recent discoveries. By trying to understand how neurons can last a lifetime, even when injured, we found that microtubules are dramatically rearranged after axon severing. Microtubule dynamics is massively upregulated and dendrite polarity switches. These changes are transcription-dependent, so we will perform transcriptome analysis to identify the microtubule regulators that are up- or down-regulated by injury. We will rapidly test candidate regulators with in vivo functional assays to identify proteis that control microtubules in normal and stressed neurons. By using a simple, genetically tractable model system that allows us to examine neurons in vivo, we will make rapid progress in understanding the basic mechanisms that control neuronal microtubule organization. We will use hypothesis-driven and discovery-driven approaches to understand microtubule regulation from multiple perspectives. As microtubules support long-term neuronal health, this work should provide a foundation for deeper understanding of pathological changes that drive neurodegenerative disease.